Description of Research Expertise

Tubes of different sizes, shapes and cellular architectures compose most organs and glands. How these tubes are formed and what factors regulate their dimensions and their pattern of branching, are among the primary questions that must be addressed before we will understand how organs are made. These questions are also of great medical relevance, because defects in tubes are at the root of many disease states, such as polycystic kidney disease and atherosclerosis, and because the recruitment of capillary tubes to tumors by sprouting angiogenesis plays a pivotal role in cancer. The long-term goal of the lab is to develop an understanding at the molecular level of the basic cell biology of tube formation and branching morphogenesis (the process by which new tubes are induced to bud and branch from pre-existing ones).

We use a simple model tubular organ (the Drosophila tracheal system) to uncover the genetic and molecular basis of how tubes are made and shaped. Using the powerful tools available to a Drosophila geneticist, we are able to manipulate gene function in individual cells and to determine the effects of such manipulations on tube morphogenesis. A large-scale forward genetic screen has been carried out and mutations in roughly 70 genes have been identified that cause striking tracheal defects (Ghabrial et al 2011).

Figure 1: Dual labeling of mosaic trachea.

Clones of homozygous cells are generated in heterozygous animals. Homozygous tracheal cells are positively marked with GFP and all tracheal cells are marked with DsRED. Here a single homozygous terminal cell (yellow) is shown. Note that a tube runs the length of each of the fine cellular processes visible in this micrograph. These tubes provide oxygen to the muscles targeted by this terminal cell.

We have focused on the problem of tube formation in tracheal terminal cells (Figure 1). These cells assume stellate shapes, similar to neurons, and build subcellular tubes that extend the length of their dendrite-like cellular processes. These seamless tubes are architecturally similar to the smallest tubes in the vertebrate vascular system. Indeed, one important project in our lab stems from the characterization of a mutation from the screen (wheezy) whose human orthologs (the Germinal Center Kinase III subfamily) are directly involved in vascular disease (Song et al 2013). We have also generated knockout flies targeting the GCKIII-binding partner CCM3, that is one of three human genes mutated in cases of familial CCM. We find that CCM3 knockout cells display the same tube dilation phenotype we described for wheezy/GCKIII. We seek to determine how cells make an internal lumen and in doing so convert cellular extensions into tubes. A related goal is to understand how lumen shape is generated and regulated. As a first step, mutants disrupting these processes (no tracheoles, cystic lumens; Figure 2) will be characterized with the aim of determining the cellular and molecular causes of the mutant phenotypes.

Figure 2: Lumen formation and shape.

From top to bottom: wild type, whacked, impatent, and cystic lumens tracheal terminal cell branches are shown. Note that the tube lumen in whacked is irregular and comes to a premature dead end, that discontinuous vacuoles fail to coalesce into a tube in impatent, and that the lumen of cystic lumens tubes shows regions of dilation suggestive of abortive side branching.

Another interest in the lab is the process of branching morphogenesis whereby cells bud from the tracheal epithelium and give rise to new tubes. Whereas terminal cell morphogenesis principally involves cells changing shape and topology, formation of larger, multicellular tubes is dependent upon cell rearrangement within the epithelium. We will focus on developing an understanding of how cells change neighbors, and of how they communicate with each other to coordinate their behaviors. Analysis of mutants that are defective in rearrangement (conjoined) and in coordinated behavior (too many leaders) will help guide our dissection of these processes.

ROTATION PROJECT:

Tube formation projects: Most organs and glands are composed of networks of branched and interconnected tubes. Some of the smallest tubes are formed within single cells by a mysterious process called “cell hollowing.” Examples of such tubes include the seamless endothelial tubes present in the mammalian vascular system as well as the terminal tubes of the Drosophila tracheal system. In a large-scale forward genetic screen carried out in Drosophila, a number of mutations that disrupt cell hollowing were identified. By determining the molecular identity of the genes affected in these mutants we aim to build a molecular understanding of how cells convert themselves into tubes.

1) Positional cloning of cystic lumens. Mutations in cystic lumens cause a striking defect in tube shape: the lumens of mutant cells show areas of constriction and dilation. To identify the genes affected by the cystic lumens mutations, a positional cloning strategy will be followed. The project will involve classical and modern genetic mapping techniques as well as molecular biology.

Branching morphogenesis projects: Formation of branched tubular organs often occurs by a process called “branching morphogenesis”. Sprouting angiogenesis in the mammalian vascular system and primary branching of the Drosophila tracheal system both employ this mode of development – in which new tubes bud from the epithelium of pre-existing tubes – to form new branches in their tubular networks. How cells rearrange within an epithelium during the formation of a new tube is not understood, although we have found that it is driven in part by a competition between cells for leading (“tip cell”) positions that divides the cells into leaders and followers (Ghabrial and Krasnow, 2006). Cells that have been sorted into the stalk of a new tube will continue to alter their arrangement in the epithelium, as they intercalate to form a longer and thinner tube. To understand the cellular and molecular mechanisms which control these morphogenetic movements we will carry out a careful phenotypic and molecular analysis of mutants that disrupt these processes.

1) Live cell imaging of re-arrangement during primary branching. Because branching morphogenesis is a dynamic process, attempting to understand the morphogenetic mechanisms at work by examination of fixed samples is problematic. By taking advantage of the genetic tools available in Drosophila, it will be possible to generate twin spot clones within the tracheal system and watch as the differently marked cells (one daughter marked with green fluorescent protein, the other marked with cherry) move relative to each other within the epithelium during primary branching. In addition, to determining how wild type control twin spots behave, we will be able to examine twin spots in which one daughter is homozygous for a mutant of interest while the other daughter is homozygous wild type.